We propose a series of studies to understand the role of bacterial viruses (vibriophage or phage) in cholera epidemiology and the evolution of Vibrio cholerae. We also hope to understand the aspects of the natural aquatic environment in a cholera endemic area that influence phage-mediated and non-phage mediated events that have biological significance to cholera. Vibriophages from Bangladesh environmental sites will be isolated and characterized. Genomic microarrays will be used to determine if they carry potential virulence genes. Phage genomes that hybridize with V. cholerae genomic DNA will be sequenced to understand their evolutionary origins. We will identify and characterize biologically active molecules produced by the growth of lytic vibriophages on V. cholerae engineered to be negative for known toxins and signaling molecules. We also will determine if phages influence the infectious dose of V. cholerae in appropriate animal models for cholera. We will test this theory that TLC is a satellite phage that carries the attachment site for the toxin- encoding CTX phage. The role of DNA transformation will be studied by looking for recombinational hot spots such as the superintegron and chromosomal islands. We will also attempt to identify factors in water that influence transformation efficiency or phage killing of V. cholerae. We will expand an established bi- weekly archive of environmental water and study variations in the water's ability to influence biological properties of V. cholerae such as biofilm formation, generation of domant cells, and stability of phages. Water will be monitored for a non-Vibrio species that acts as an alternative host for vibriophages. Such a host will be targeted for environmental surveillance in order to assess its potential role as a co-factor in promoting vibriophage "blooms" within aquatic environments which in turn should have a deleterious effect on the concentration of phage sensitive epidemic V. cholerae strains. The seasonal fluctuation of the parameters we selected may be strong predictors of cholera seasonality in Bangladesh and thus aid in the control of this deadly disease by a variety of interventions.